Bio‐functional hydrogel with antibacterial and anti‐inflammatory dual properties to combat with burn wound infection

Abstract Burn infection delays wound healing and increases the burn patient mortality. Consequently, a new dressing with antibacterial and anti‐inflammatory dual properties is urgently required for wound healing. In this study, we propose a combination of methacrylate gelatin (GelMA) hydrogel system with silver nanoparticles embed in γ‐cyclodextrin metal–organic frameworks (Ag@MOF) and hyaluronic acid‐epigallocatechin gallate (HA‐E) for the burn wound infection treatment. Ag@MOF is used as an antibacterial agent and epigallocatechin gallate (EGCG) has exhibited biological properties of anti‐inflammation and antibacterial. The GelMA/HA‐E/Ag@MOF hydrogel enjoys suitable physical properties and sustained release of Ag+. Meanwhile, the hydrogel has excellent biocompatibility and could promote macrophage polarization from M1 to M2. In vivo wound healing evaluations further demonstrate that the GelMA/HA‐E/Ag@MOF hydrogel reduces the number of the bacterium efficiently, accelerates wound healing, promotes early angiogenesis, and regulates immune reaction. A further evaluation indicates that the noncanonical Wnt signal pathway is significantly activated in the GelMA/HA‐E/Ag@MOF hydrogel treated group. In conclusion, the GelMA/HA‐E/Ag@MOF hydrogel could serve as a promising multifunctional dressing for the burn wound healing.


| INTRODUCTION
Burn injury causes estimated 265,000 deaths every year, 1 and burn wound infection would lead to the increasing mortality of the hospitalized burn patients. Because of the complex microenvironment, burn wounds are colonized with bacteria, which secrete much exudate and delay wound healing 2,3 compared to other forms of trauma. Besides, the inflammation from the polarization of the macrophages also plays a critical role, which could kill potential pathogens by contributing to the necessary inflammation, and when the pathogens are once cleared, the inflammation will be resolved and then the tissue remodeling and regeneration will be initiated. 4 Currently, artificial dermal substitutes and dressing management are the main therapeutic methods for the burn wound excision. 5 However, current commercial antibacterial dressing lacks appropriate bioactivity to regulate the complex microenvironment of the burn wound, such as the polarization of the macrophages. Therefore, new antibacterial materials have great clinical value to satisfy the aforementioned demands. 6 The use frequency of the traditional antibiotics has decreased owing to the continuous emergency of the multidrug-resistant bacteria. Silver nanoparticles (Ag-NPs) are extensively used as antibacterial drugs in cutaneous wound repair 7 because of the good antibacterial activity and little vulnerability to the bacterial resistance. However, a silver cations (Ag + ) burst of the Ag-NPs may increase the cytotoxicity. 8,9 In addition, small Ag-NPs have a deficiency in stability because of the cluster aggregations. Cyclodextrin metal-organic framework (CD-MOF) is a framework constructed by an organic ligand, cyclodextrin, with high porosity and a large specific surface area. Therefore, Ag-NPs embedded in metal-organic frameworks (Ag@MOF) can make Ag-NPs keep stable and be released gradually, which is an effective treatment for diabetic wounds. 10 Prior study reveals that a hydrogel, as a drug delivery system, could allow various biomaterials to realize a controlled and sustainable release. 11 Gelatin methacryloyl (GelMA) has the same biological properties as the natural extracellular matrix (ECM), which enhance the cell spread and proliferation. 6,12 Therefore, GelMA hydrogel could realize the sustained release of Ag@MOF. 13 Infections in burn wounds may contribute to a local inflammatory response, an angiogenesis defect and an up-regulated expression of the pro-inflammation cytokines. Epigallocatechin gallate (EGCG) exerts numerous biological functions, 14,15 such as antibacterial activity, freeradicals scavenger, regulation of the inflammation, wound healing and skin regeneration, 16 as the major catechin in green tea. Moreover, EGCG could reduce the expression of the pro-inflammatory factors in lipopolysaccharide (LPS)-induced macrophages in vitro and elevate the antiinflammatory cytokine IL-4, 17 which indicates that EGCG could possibly induce the polarization of macrophages toward an anti-inflammation phenotype. However, owing to the 1,2,3-trihydroxyphenyl moieties in B and D ring, 18 EGCG is susceptible to the oxidation. 19,20 The hyaluronic acids (HA) are known to have anti-oxidant activity, 21 and hence conjugating the HA onto EGCG to form HA-E polymer is a feasible approach, which could reduce the rate of oxidation and remain the biological effects of EGCG 18 by maintaining these sites during the chemical reactions.
To satisfy all the desired properties of a hydrogel for burn wound infection healing, we design a hydrogel system to minimize the bacterial infections at the wound (through the use of Ag@MOF), promote vascularization, regulate inflammatory (through the use of HA-E), and finally accelerate the healing of wounds (Scheme 1). The polyphenolic groups of EGCG are reacted with aldehydes of 2-diethoxyethylamine (DA) by Baeyer acid to form EGCG dimer, and then the tyramines of EGCG dimers are grafted to the carboxyl groups on HA by EDC/NHS chemistry. Then the physical properties, the cell compatibility and the antibacterial and anti-inflammatory effects of the hydrogel are employed in vitro. 22 Next, we further investigate the antibacterial, anti-inflammation and wound healing effects in full-thickness burn infection. Finally, the potential mechanism of the prepared hydrogels in facilitating burn wound healing is investigated for the further study.

| Synthesis of Ag@MOF
The CD-MOF crystals were synthesized according to the previously published methods. 23 Briefly, 97.3 g γ-CD (biotech grade, Macklin, China) and 33.6 g KOH (90%, Macklin) were dissolved in 3 L of deionized water (DIW). After being filtrated by 0.45 μm filtration, 1.8 L of methanol (analytical grade, Sinopharm Chemical Company, China) was evaporated at 50 C for 20 min and the methanol vapor was gradually diffused into DIW. In order to trigger the crystallization, after adding 38.4 g of polyethylene glycol (PEG) (Mw = 20,000, Macklin) and a further 10 min stirring, the obtained mixture was placed overnight at ambient temperature. Finally, these CD-MOF crystals were rinsed with anhydrous ethanol and dried overnight to collect the crystals.
The Ag@MOF was synthesized as the previously established protocols. 24 Briefly, 600 mg of CD-MOF crystals was dissolved in 1.5 ml of acetonitrile (analytical grade, Sinopharm Chemical Reagent Company, China), and then 5 ml AgNO 3 precursor (20 mmol/L, Sinopharm Chemical Reagent Company) was added. The solution was centrifuged after a 72 h's reaction, and the residue was rinsed by acetonitrile for several times, and then lyophilized for further use.

| Synthesis of GelMA and HA-E
Depending on the reported protocols, GelMA was synthesized with slight modifications. 25 First, 20 g of gelatin (Sigma Aldrich Corporation, USA) was fully dissolved in 250 ml DIW at 60 C. Then methacrylic anhydride (94%, Macklin) was added in the solution drop by drop at a volume of 0.6 ml/(gram gelatin). The solution was transferred into a dialysis bag, which was sank in DIW for 3-5 days at room temperature (MwCO = 8000) after reacting at ambient temperature for 8 h. Ultimately, to obtain GelMA, the collected liquid was centrifuged, filtrated by neural filter and lyophilized. HA-E was synthesized by following a previous report. 26

| Preparation of composite hydrogels
In order to prepare the hydrogel, GelMA was fixed to 10 wt% in DIW and then adding various concentrations of HA-E (1, 1.5, and 2 wt%) in the solution. Then, LAP (0.1 wt%, Yinchang New Material Co., Ltd., China) was added as a photoinitiator. The final mixture was added into a 48-well plate, and then exposed to UV light (365 nm) for 10 s at the power density of 10 mW/cm 2 . For preparation of GelMA/HA-E/Ag@MOF hydrogel, Ag@MOF was dispersed in GelMA (10 wt%) solutions with different concentrations of 20, 40, and 80 μg/ml, respectively. Then, then adding HA-E (1 wt%) solution to prepare the mixed solutions. The prepared GelMA/HA-E/Ag@MOF(10), GelMA/HA-E/Ag@MOF (20) and GelMA/HA-E/ Ag@MOF(40) hydrogels were also in accordance with the above procedure. 2.5 | Physical evaluation of the composite hydrogels 2.5.1 | Swelling ratio of the hydrogels According to a gravimetric method previously reported, the swelling ratio of the hydrogels was tested and then measured. 24,28 Briefly, the hydrogel samples were weighed, which was recorded as W 0 and then completely sank into phosphate-buffered saline (PBS, GIBCO, USA) at 37 C. The soaked hydrogels were removed from the PBS, the moisture on the hydrogel was gently absorbed by filter paper, and the swollen weight (W t ) was noted down immediately at given time intervals (1 h, 2, 3, 4, 5 and 6 h). The following equation was used for the calculation of the swelling ratios of the hydrogels:
For determining the linear viscoelastic region of the hydrogels, conducting dynamic strain scanning from 0.1 to 10 rad/s À1 in ambient conditions and the storage modulus (G') and loss modulus (G") were obtained and then carefully measured.

| Compression test
The obtained stress/strain curve was measured through a TA rheometer instrument (AR 1500Ex; TA Instrument, USA) to extract the compressive modulus of each hydrogel. In the test, the hydrogels were placed between two compression plates and were compressed using a flat probe at 0.05 mm/s to 60%.

| Degradation in vitro
The enzymatic degradation experiments were used to measure the biodegradation performance of hydrogels. Briefly, the hydrogel samples were soaked in PBS consisting of 0 or 100 U/ml hyaluronidase, respectively, at 37 C at 70 rpm. At certain time intervals, the hydrogels were taken out, followed by deliberate procedure including washing, freeze-drying and weighing. To illustrate the biodegradability, the degradation ratio was computed with the following equation: where W 0 is the original dry weight after lyophilization, and W t is the dry weight after lyophilization at a designed time.

| Release of Ag +
The ICP-MS method was used to detect the release of Ag + from GelMA/HA-E/Ag@MOF hydrogels. Briefly, GelMA/HA-E/Ag@MOF hydrogels (600 μl) was added to 10 ml of PBS. At certain time (1, 2, 3, 4, 5, and 7 days), all PBS supernatant liquor was collected and then the same amount of fresh PBS were replenished instead. Lastly, the collected samples were digested with nitric acid to determine the concentrations of Ag + ions.
Briefly, the hydrogel sample (600 μl) was added in 24-well plates and 1.8 ml mixed bacterial suspension at a final concentration of 1 Â 10 8 CFU/ml was mixed together. Following co-culturing at 37 C for 1 day, the mixture was diluted serially, and 100 μl of each dilution was cultured at 37 C on LB agar plates. The following equation was used for the calculation of the antibacterial ratio (AR) of the hydrogels: where N control is the number of colonies in GelMA hydrogel and the N sample is the number of colonies in GelMA/HA-E or GelMA/HA-E/ Ag@MOF hydrogel.

| Western blot of wound tissues
For western blot analysis for tissues, the skin samples excised from rats in each group were completely homogenized with proteinase-containing protein extraction reagent for 10 times.
After centrifuged at 10,000 rpm for 20 min at 4 C, the supernatant was collected to another tube so as to perform a quantitative analysis by protein assay kit. The proteins (50 μg) were loaded and heated to 95 C for 10 min followed by other experimental steps as described previously.

| Statistical analysis
All experiments in this study performed were repeated at least three times with three wells repeated per treatment. The collected data was performed as means ± SD. Utilizing one-way analysis variance (ANOVA) analysis by SPSS 25 (IBM). A p value of <0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

| Characterization of nanoparticles and hydrogel
The SEM image demonstrated that the obtained CD-MOF appeared as individual regular crystals and had a uniform size around 143 nm ( Figure 1a), because of the organic framework linked by γ-cyclodextrins (γ-CDs) and alkali metal salts. The TEM image indicated that the synthesized Ag@MOF had a spherical shape with a small size (Figure 1b). The was successfully grafted onto gelatin. 29 Additionally, a characteristic resonance of EGCG (δ = 6.99, 6.62, and 6.15 ppm) was observed in HA-E (Figure 1e), assigning to the hydrogen peaks of the benzene ring in EGCG, which verified the successful preparation of HA-E. 30 Moreover, such chemical synthesis of HA-E can maintain the biological function of EGCG, which mainly derived from its function groups. 18 Further, FTIR demonstrated Gel-, HA-, MA-, and EGCG-related peaks at around 1200 cm À1 (Figure S2), indicating that our results were consistent with the previous references [31]. Figure S3A showed the 114.5% ± 8.6%), which was slightly higher than that of GelMA hydrogel (33.3% ± 0.8%). These results indicated that the addition of 1% HA-E may maintained the excellent structural stability without notable swelling.

| Rheological and compression properties
Referring to the previous study, the viscoelastic properties of the hydrogels were used to assay the stability of the cross-linked networks. 37

| Ag + release studies and in vitro degradation
The EDS spectrum showed that Ag was coexisted with other elements (Figure 2f The weight loss curves of the hydrogels showed a gradual decrease in Figure S5A, but there was a sharp dropping rate in lysozyme group ( Figure S5B). Compared with pure GelMA hydrogel (44.4% ± 1.4%, 65.3% ± 2.1%) at the fifth day in the presence and absence of lysozyme, the degradation rate was relatively sharp as the increasing of the concentration of HA-E, ranging from 49.9% ± 2.0% to 65.7% ± 1.6% and 67.2% ± 1.6% to 84.3% ± 1.7% respectively. 38 Both GelMA and HA-E have the sequence to react with lysozyme. 38,39 When the hydrogel is degraded, it is advantageous for the loaded material to be released from the hydrogel in a sustainable speed and fully cleared from it to accumulate in the intended site. 40 Hence, the appropriate degradation rate could not only maintain the stability of the hydrogel but also promote the drug consistently release. were reduced to 75.3% ± 0.8% for E. coli, 88.8% ± 1.3% for S. aureus, and 82.1% ± 1.4% for P. aeruginosa. In addition, all hydrogels loaded with Ag-NPs (GelMA/HA-E/Ag@MOF) exhibited much better antibacterial property with the antibacterial ratio of >95%, which increased to 100% when Ag@MOF was 40 μg/ml. Prior studies reported that the Ag-NPs and EGCG had antibacterial property, which were verified by the results. 41 Considering the biocompatibility and the antibacterial ratio, 20 μg/ml of Ag@MOF was selected for the following experiment.

| Biocompatibility of the hydrogels
The biocompatibility of the prepared hydrogels was assessed using 3 T3 cells through CCK-8, live/dead staining and F-actin staining. 36 The live/dead staining results revealed higher density of the viable cells in the GelMA/HA-E hydrogel group than those in the other two groups with few dead cells (red) present and normal cell morphology ( Figure 4a). Resembling the prior live/dead staining results, the cytoskeleton architecture of 3T3 cells in all groups showed that the cells were spindle-shaped morphology during the experiment (Figure 4b).
Moreover, GelMA/HA-E group showed a number of cell-cell contacts at the fifth day, which formed an interconnected network. These

| In vitro polarization of macrophages
Macrophages were first classified into M1 macrophages (a proinflammatory phenotype) and M2 macrophages (an antiinflammatory phenotype). 42 The hydrogel with the ability to facilitate the M1-to-M2 transition of the macrophages would be favorable for the wound healing. In Figure Figure 5a displayed the process of injecting the hydrogels on the infectious burn wound, which was the same injectable as previous study. 7 As shown in Figure 5b,c, wound area at the Day 0 was recorded and measured as initial area (100%).
Compared with others group (86.99% ± 1.9%, 62.66% ± 0.7%, 64.47% ± 1.2%, and 65.47% ± 2.4%), the wound area (%) in GelMA/ HA-E/Ag@MOF group (51.98% ± 2.5%) was significantly smaller and healed better at the third day after the surgery. When this tendency continued until the seventh day, between GelMA/HA-E/Ag@MOF group (18.95% ± 0.6%) and the gauze group (61.87% ± 0.8%), it was shown a significant difference in wound size. At the 10th day, GelMA/HA-E/Ag@MOF group showed the smallest wound area (2.47% ± 0.2%) among the groups, which was basically healed, while the other groups with approximate 6% left. At the 14th day, there were no obvious wound could be seen in all groups, which indicated the epidermis was almost closed.
To further explore the therapeutic efficacy of the hydrogel on the regeneration of epidermis and dermis from a microscopic point of view, histological analysis such as hematoxylin and eosin (HE) and Masson staining were performed. 44 The closure of the wound was measured by calculating the gap of the wound in HE staining images ( Figure 5d). Since the wound tissues were difficult to calculate at the third day, Figure 5e

| Histological analysis
The wound healing process can be subdivided into four processes: hemostasis, inflammation, proliferation, and remodulation. 46 The inflammatory phase serves to prevent infection and activate signals required for the wound repair. Besides the infection, the burn wound also suffers from the uncontrolled immune response and hyperexuberant cytokine production during the inflammatory phase (including hemostasis and inflammation). 46 In the proliferative phase, a new blood vessel-network was constructed to supply the newly formed granulation tissue, which could bring oxygen and nutrients to the wound bed. 47 Severe burn wounds lose much dermal blood flow and hence neovascularization plays a key role in the wound healing. 48 The HE staining was used to assay the morphology and area of the microvessels in the wound section. The microvessels were clearly seen in all

| In vivo effect of hydrogel on macrophages polarization and inflammation microenvironment
Macrophages play a crucial role in various physiological and pathological processes in early wound infections. 51 As widely distributed innate immune cells, pro-inflammatory phenotype macrophages effectively defend against pathogens and promote wound healing.
Following successful resuscitation, burn wound then face the and produce a wide array of the inflammatory cytokines (i.e., TNFα), which induce tissue destruction and healing delay. Besides, the inflammation phase in wounds should transition into the proliferation phase in the following steps. Macrophage infiltration is vital step in the wound healing process. 52 When the number of M2 phenotype macrophages is significantly increased, which will produce high expressions of anti-inflammatory cytokines (i.e., TGF-β1) to reduce the macrophages infiltration, accelerate tissue repair, and generate a favorable anti-inflammatory microenvironment. 53 If macrophages could not successfully prompt macrophages to shift toward the two phenotype, which will increase secretion of foreign body giant cell (FBGC) formation and fibrous enhancer factor, resulting in the healing delay. 54 As previously proved in vitro, GelMA/HA-E/Ag@MOF hydrogel could facilitate M2 polarization of the macrophages, we inferred that whether the material could also play an inflammation regulating role in vivo. To characterize the phenotype of the macrophage cells at the seventh day, we stained the tissue cells with CD68, CRR7 (M1 marker), and CD163 (M2 marker) antibodies by immunohistochemistry staining. As shown in Figure 8a

| CONCLUSIONS
In summary, we successfully prepared HA-E and Ag@MOF-loaded GelMA hydrogels for the treatment of burn wound infections in rats.
By adjusting the concentration of HA-E, the excellent pore-size distribution, appropriate physical properties and antibacterial activity make writingreview and editing (lead).